EP2619584B1 - Nouveau procédé pour diagnostiquer la maladie de lyme en utilisant un test immunologique cellulaire - Google Patents

Nouveau procédé pour diagnostiquer la maladie de lyme en utilisant un test immunologique cellulaire Download PDF

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EP2619584B1
EP2619584B1 EP11764357.7A EP11764357A EP2619584B1 EP 2619584 B1 EP2619584 B1 EP 2619584B1 EP 11764357 A EP11764357 A EP 11764357A EP 2619584 B1 EP2619584 B1 EP 2619584B1
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pro
inflammatory cytokine
borrelia
expression level
sample
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EP2619584A1 (fr
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Leonardus Antonius Bernardus Joosten
Mihai Gheorghe Netea
Johannes Willem Maarten Van Der Meer
Bart Julian Kullberg
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Stichting Katholieke Universiteit
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6866—Interferon
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6869—Interleukin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/20—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Spirochaetales (O), e.g. Treponema, Leptospira
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a method for diagnosing Lyme disease in a subject, using the determination of a pro-inflammatory cytokine expression level.
  • Lyme disease or Lyme borreliosis is a vector-borne, multisystem inflammatory bacterial illness transmitted to humans by the bite of ticks ( Ixodes species) carrying spirochetes of the genius Borrelia.
  • the disease presentation varies widely, and may include rash and flu-like symptoms in its initial stage, and musculoskeletal, arthritic, neurologic, psychiatric and cardiac manifestations in later stages. Lyme disease is clinically manifested in three phases as:
  • PCR Polymerase chain reaction
  • the invention relates to a method for diagnosing Lyme disease in a subject, the method comprising the steps of:
  • diagnosis Lyme disease preferably means that a diagnosis is reached in at least the following cases presented below:
  • “early stage” preferably means shortly after the clinical apparition of symptoms of local skin inflammation (EM) or before the stage wherein the disease has disseminated into the heart and/or nervous system or early disseminated into the heart and/or nervous system and/or before the stage of a disseminated disease involving featuring motor and sensory nerve damage and brain inflammation and arthritis also called a late disseminated disease involving featuring motor and sensory nerve damage and brain inflammation and arthritis as defined later herein.
  • EM local skin inflammation
  • the method of the invention could provide a specific diagnostic of the Lyme disease at an early stage in these cases.
  • "early stage” preferably means before the stage wherein the disease has disseminated into the heart and/or nervous system or early disseminated into the heart and/or nervous system and/or before the stage of a disseminated disease involving featuring motor and sensory nerve damage and brain inflammation and arthritis also called a late disseminated disease involving featuring motor and sensory nerve damage and brain inflammation and arthritis as defined later herein.
  • the method of the invention also provides a specific diagnostic of the Lyme disease, preferably at an early stage preferably before the stage wherein the disease has disseminated into the heart and/or nervous system or early disseminated into the heart and/or nervous system and/or before the stage of a disseminated disease involving featuring motor and sensory nerve damage and brain inflammation and arthritis also called a late disseminated disease involving featuring motor and sensory nerve damage and brain inflammation and arthritis as defined later herein.
  • the disease may also be diagnosed using a method of the invention in a later stage in case the patient will consult a physician in a later stage.
  • a later stage is preferably before the stage wherein the disease has disseminated into the heart and/or nervous system or early disseminated into the heart and/or nervous system and/or before the stage of a disseminated disease involving featuring motor and sensory nerve damage and brain inflammation and arthritis also called a late disseminated disease involving featuring motor and sensory nerve damage and brain inflammation and arthritis as defined later herein.
  • the method of the invention is also attractive compared to classical methods since at this later stage classical methods may not give a positive response or may give a positive response. However, classical methods often give false positive responses.
  • the disease may be diagnosed when the disease has disseminated into the heart and/or nervous system and/or at the stage of a disseminated disease involving nerve damage, brain inflammation, or arthritis also called a late disseminated disease involving nerve damage, brain inflammation, or arthritis and/or at the stage wherein the disease may become persistent or refractory to a 2 weeks antibiotic treatment.
  • the clinical apparition of symptoms of local skin inflammation may be assessed by the physician. Local skin inflammation is usually characterized by an Erythema migrans (EM) that occurs at the site of the tick bite.
  • EM Erythema migrans
  • the rash is usually salmon to red-colored; the color may cover the entire lesion or may have an area in the centre that is flesh-colored.
  • the rash consists of multiple rings, which give it a "bull's eye” appearance.
  • the invention relates to a method for diagnosing Lyme disease shortly after the clinical apparition of symptoms of local skin inflammation (EM) in an subject, the method comprising the steps of:
  • the assessment that the disease has already reached a stage wherein it has disseminated into the heart and/or nervous system may be assessed using serology, which has a poor sensitivity and specificity.
  • the assessment that the disease has already reached a disseminated stage with, among others, featuring motor and sensory nerve damage and brain inflammation and arthritis, also called a late disseminated stage with, among others, featuring motor and sensory nerve damage and brain inflammation and arthritis may be assessed using PCR to detect bacterial DNA in these organs/tissues.
  • organ biopsies are very seldom available for testing in patients with chronic Lyme disease.
  • shortly after the clinical apparition of symptoms of local skin inflammation preferably means at least one day, at least two days, at least three days, at least four days, at least five days, at least six days, at least seven days, at least eight days, at least nine days, at least ten days at least 15 days, at least 20 days, at least 25 days, at least 30 days or more after the clinical apparition of symptoms of local skin inflammation.
  • EM local skin inflammation
  • "before” preferably means at least one day, at least two days, at least three days, at least four days, at least five days, at least six days, at least seven days, at least eight days, at least nine days, at least ten days at least 15 days, at least 20 days, at least 25 days, at least 30 days or more before the stage wherein the disease has disseminated into the heart and/or nervous system or early disseminated into the heart and/or nervous system and/or before the stage of a disseminated disease involving featuring motor and sensory nerve damage and brain inflammation and arthritis also called a late disseminated disease involving featuring motor and sensory nerve damage and brain inflammation and arthritis.
  • diagnosis preferably means a predictive risk assessment of the subsequent development of Lyme disease in a subject.
  • a subject may be an animal or a human being.
  • the diagnosis method may be applied as often as necessary in a subject.
  • a subject diagnosed is a subject suspected to have a high risk of having or developing chronic Lyme disease, due for example to the fact that this subject lives in a region wherein the Lyme disease is common, this subject spends a lot of time outdoors, including a subject who works outdoors, gardens, or participates in outdoor activities such as hunting or hiking or subject has a pet.
  • a subject is a human being.
  • the Lyme disease is diagnosed when step (c) leads to the finding of a detectable expression level or an increase of the expression level of a pro-inflammatory cytokine.
  • a pro-inflammatory cytokine is a cytokine that is able to promote systemic inflammation.
  • a pro-inflammatory cytokine is IL-1 ⁇ .
  • Other pro-inflammatory cytokines according to the disclosure are selected from the group consisting of IFN ⁇ , IL-6, and IL-17. Good results were obtained using IL-1 ⁇ . IL-1 ⁇ is therefore a preferred pro-inflammatory cytokine in this context.
  • a reference value for the expression level of a pro-inflammatory cytokine is preferably the average value for said expression level in a control sample.
  • a control sample is derived from a control subject or from control subjects or from the medium or culture medium used for step (b).
  • a control subject may be a subject who do not live in a region at risk or who does not spend a lot of time outdoors.
  • a pro-inflammatory cytokine tested may be not detectable in "a reference value”. Said reference value may therefore be 0 or nor detectable in an assay as defined herein. In other words, said cytokine may not be detectable in a control sample.
  • the assessment of the expression level of the respective pro-inflammatory cytokine may be directly realised at the protein expression level (quantifying the amount of the cytokine), and/or indirectly by quantifying the amount of a nucleotide sequence encoding the respective cytokine (the value from a subject wherein the method is being carried out and optionally the reference value from a control sample).
  • a nucleotide acid sequence encoding a human IL-1 ⁇ , IFN ⁇ , IL-6, IL-17 is given as SEQ ID NO:1, 2, 3, 4 respectively.
  • a corresponding amino acid sequence of human IL-1 ⁇ , IFN ⁇ , IL-6, IL-17 is given as SEQ ID NO:5, 6, 7, 8 respectively.
  • a pro-inflammatory cytokine to be quantified has:
  • a nucleotide sequence encoding a pro-inflammatory cytokine means a messenger RNA (mRNA).
  • mRNA messenger RNA
  • the expression level of a pro-inflammatory cytokine is determined directly by quantifying the amount of said pro-inflammatory cytokine.
  • Quantifying a polypeptide amount may be carried out by any known technique.
  • a polypeptide amount is quantified using a molecule which specifically binds to said pro-inflammatory cytokine.
  • Preferred binding molecules are selected from: an antibody, which has been specifically raised for recognizing said pro-inflammatory cytokine, any other molecule which is known to specifically bind said pro-inflammatory cytokine.
  • Such antibody could be used in any immunoassay known to the skilled person such as western blotting, or ELISA (Enzyme-Linked Immuno Sorbent Assay) or FACS (Fluorescence Activated Cell Sorting) using latex beads.
  • the preparation of an antibody is known to those skilled in the art. A short explanation of methods that could be used to prepare antibodies is later herein given. Suitable antibodies are commercially available. For example antibodies from R&D systems could be used to assess IL-1 ⁇ , IL-6, IL-17 or IFN ⁇ .
  • any other molecule known to bind the cytokine tested may be a nucleic acid, e.g. a DNA regulatory region, a polypeptide, a metabolite, a substrate, a regulatory element, a structural component, a chaperone (transport) molecule, a peptide mimetic, a non-peptide mimetic, or any other type of ligand. Mimetic is later herein defined.
  • molecules known to bind a pro-inflammatory cytokine include a receptor for said pro-inflammatory cytokine, an antibody directed against said pro-inflammatory cytokine.
  • IL-1 ⁇ is chosen as a pro-inflammatory cytokine
  • a preferred anti-IL1 ⁇ antibody is the IL-1F2 (from R&D systems) antibody.
  • Binding of a pro-inflammatory cytokine to a second binding molecule may be detected by any standard methods known to those skilled in the art. Suitable methods include affinity chromatography co-electrophoresis (ACE) assays and ELISA.
  • ACE affinity chromatography co-electrophoresis
  • the quantification of a substrate of a corresponding polypeptide or of any compound known to be associated with a function or activity of a corresponding polypeptide or the quantification of a function or activity of a corresponding polypeptide using a specific assay is encompassed within the scope of the diagnosis method of the invention.
  • trans-activation of a target gene of a pro-inflammatory cytokine or a molecule known to bind a pro-inflammatory cytokine can be determined and quantified, e.g., in a transient transfection assay in which the promoter of the target gene is linked to a reporter gene, e.g., P-galactosidase or luciferase.
  • a reporter gene e.g., P-galactosidase or luciferase.
  • a method of the invention may encompass determining the expression level of at least one, or two or three or four pro-inflammatory cytokines in a sample at the end of step (b). For example IL-1 ⁇ and IL-6 or IFN ⁇ and IL-17 could be assessed. It is expected that IL-1 ⁇ and IL-6 are produced within 48 hours of culture, whereas IFN ⁇ and IL-17 are expected to be produced within 7 days of culture.
  • a sample from a subject is used.
  • a method of the invention is therefore an in vitro or ex vivo method.
  • a sample is blood, is diluted blood, or is derived from blood.
  • Disclosed samples preferably comprise or consist of a fluid obtained from a subject. More preferably, a disclosed fluid comprises or consists of or is selected from: urine, spinal cord fluid, saliva, semen, or bronchoalveolar lavage. According to the invention, the preferred fluid is, comprises, consists of or is derived from blood. Blood may be diluted before being further used. The dilution may be 1:4, 1:5 or 1:6. The dilution is preferably carried out in a medium, preferably a culture medium such as RPMI 1640 or a buffered solution.
  • said obtained sample of step (a) is subsequently contacted with a source of Borrelia antigens.
  • the contacting step may have a duration of at least 1, 2, 3, 4, 5, 6, 7, 8 up to 24 hours. Disclosed contacting steps can be longer. Preferably the contact has a duration of 4-24 hours. Disclosed contacting steps are of 4-96 hours.
  • This contact step may be a culture step in a culture medium such as RPMI 1640. At least 10 4 and up to 10 6 bacteria may be inoculated at the start of the contacting step.
  • Preferred species of Borrelia as a source of Borrelia antigens include: B. burgdorferi, more preferably the strain ATCC 35210, B.
  • the Borrelia antigen is preferably from a species of Borrelia selected from: B. burgdorferi, B. garinii and B. afzelii .These strains are preferred since they are the most widely present in Europe and in America.
  • a source of a Borrelia antigen may mean that a whole Borrelia cell or a Borrelia cell is being used.
  • a whole Borrelia cell or a Borrelia cell is heat-inactivated or formalin fixated. Heat-inactivated could be replaced by heat-killed.
  • Heat-inactivated cells are preferably prepared by heating at 52, 53, 54, 55 or 56 for 20, 25 or 30 minutes. More preferably heat-inactivated cells are prepared by heating at 52°C for 30 minutes.
  • Formalin fixated cells may be obtained by contacting the cells with formaldehyde for 40, 50, 60 minutes.
  • part of a Borrelia cell may be used.
  • a part of a Borrelia cell is preferably an antigenic part thereof; it comprises or consists of an antigen.
  • An antigen may be a protein, a digest of the protein and/or a fragment thereof, which may be in a purified form or may be comprised within a crude composition, preferably of biological origin, such as a lysate, sonicate or fixate of a Borrelia.
  • an antigen may be chemically synthesized or enzymatically produced in vitro.
  • the source of a protein, or fragment thereof as antigen may also be a nucleic acid encoding said, or fragment thereof, from an RNA or DNA template.
  • a source of a Borrelia antigen is a whole Borrelia cell or an antigen from said cell.
  • the use of a whole Borrelia cell is attractive and preferred above the use of a part of a cell for at least two reasons. The use of a whole cell is easier and cheaper for the skilled person. There is no need to identify and subsequently synthesize suitable parts (i.e. antigenic parts) of a cell. In addition, by using a whole cell, all potential suitable part (i.e. all antigens) of said cell are present.
  • the diagnostic method is therefore expected to be far more sensitive and efficient than a corresponding diagnostic method carried out using a given antigen.
  • step (b) In a preferred method wherein in step (b) several species of Borrelia are used as a source of a Borrelia antigens, the sample of step (a) is divided into several sub-samples, each sub-sample being contacted with a source of antigens from one species of Borrelia.
  • This preferred method may allow us to identify the Borrelia species that infected a given subject.
  • a pro-inflammatory cytokine which is IL-1 ⁇
  • the expression level of a pro-inflammatory cytokine is determined in said sample at the end of step (b).
  • a nucleotide sequence encoding said pro-inflammatory cytokine and/or said pro-inflammatory cytokine are extracted and optionally purified using known methods to the skilled person.
  • the supernatant is isolated by centrifugation. Centrifugation may be carried out at 1200 rpm and at 4°C.
  • a detergent to the sample at the end of step b). Several detergents could be used such as Triton X 0.1 %. Adding a detergent is attractive since it is expected that no centrifugation step is needed.
  • Lyme disease is diagnosed when the expression of a pro-inflammatory cytokine is detectable or detected and optionally when the comparison leads to the finding of a detectable expression of said pro-inflammatory cytokine and/or an increase of the expression level of said pro-inflammatory cytokine.
  • pro-inflammatory cytokine expression is generally not detectable.
  • Detection of the expression of a pro-inflammatory cytokine or an increase of the expression level of said pro-inflammatory cytokine and/or an increase or a detection of the expression level of a nucleotide sequence encoding said pro-inflammatory cytokine (or steady state level of said pro-inflammatory cytokine) is preferably defined as being a detectable expression level or a detectable change of the expression level of said pro-inflammatory cytokine and/or of a nucleotide sequence encoding said pro-inflammatory cytokine (or steady state level of the encoded pro-inflammatory cytokine or any detectable activity of said pro-inflammatory cytokine or detectable change in a biological activity of said pro-inflammatory cytokine) using a method as defined earlier on as compared to the expression level of said pro-inflammatory cytokine and/or of a corresponding nucleotide sequence (or steady state level of the corresponding encoded pro-inflammatory cytokine) in a control subject or in a control.
  • an increase of the expression level of a nucleotide sequence encoding a pro-inflammatory cytokine means an increase of at least 5% of the expression level of said nucleotide sequence using PCR.
  • Preferred primers used for the PCR are identified as SEQ ID NO:9 and SEQ ID NO:10 when a pro-inflammatory cytokine is IL-1 ⁇ : forward CAGCTACGAATCTCCGACCAC and reverse GGCAGGGAACCAGCATCTTC.
  • an increase of the expression level of a nucleotide sequence means an increase of at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, at least 150%, or more.
  • an increase of the expression level of a pro-inflammatory cytokine means an increase of at least 5% of the expression level of said pro-inflammatory cytokine using western blotting and/or using ELISA or a suitable assay. More preferably, an increase of the expression level of a polypeptide means an increase of at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, at least 150%, or more.
  • an increase of a pro-inflammatory cytokine activity means an increase of at least 5% of the polypeptide activity using a suitable assay. More preferably, an increase of the polypeptide activity means an increase of at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, at least 150% or more.
  • Lyme disease is diagnosed when the detection or comparison leads to the finding of a detectable level or an increase of the level of expression of a pro-inflammatory cytokine or an increase or a detection of the expression level of a nucleotide sequence encoding said pro-inflammatory cytokine, said detection or increase being detected at the level of the amino acid sequence of said pro-inflammatory cytokine, more preferably an increase of at least 5% of the expression level of said pro-inflammatory cytokine using ELISA as defined herein.
  • the method of the invention is attractive since the diagnosis is reached early enough in order to treat a diagnosed subject to prevent damage. Furthermore, this method is non-invasive, simple, reproducible, sensitive, specific, and time and cost efficient.
  • an assay device for diagnosing Lyme disease in a subject, wherein said device comprises a molecule which specifically binds pro-inflammatory cytokine IL-1 ⁇ .
  • This device may be used in a diagnosis method of the invention. Any subject or physician could use this device at office/home, repeat the use of such device as often as necessary.
  • a molecule which specifically binds a pro-inflammatory cytokine and which is present in the device is an antibody.
  • an assay device is a lateral flow test strip also known as dipstick, preferably, though not necessarily, encased in a housing, designed to be read by the subject, and the assay is a sandwich immunoassay.
  • Such devices are impregnated with reagents that specifically indicate the presence of a given molecule, here a cytokine by changing colour upon contact with a sample.
  • a cytokine by changing colour upon contact with a sample.
  • Preferred subject's samples have already been defined herein.
  • An antibody is preferably labelled by conjugation to a physically detectable label, and upon contacting with a sample containing a pro-inflammatory cytokine forms a complex.
  • Said antibody-pro-inflammatory cytokine complex is then contacted with a second antibody, which recognizes said first antibody and which is immobilized on a solid support within the device.
  • a second antibody captures said antibody-pro-inflammatory cytokine complex to form an antibody-pro-inflammatory cytokine-antibody sandwich complex, and the resulting complex, which is immobilized on the solid support, is detectable by virtue of the label.
  • a test strip may then be inserted into a reader, where a signal from said label in the complex is measured. Alternatively, a test strip could be inserted into the reader prior to addition of the sample.
  • the presence of a pro-inflammatory cytokine is visualised by a subject as a change of colour of at least part of a device.
  • Dipsticks are usually made of paper or cardboard.
  • additional molecules are present in a device as a positive or negative control.
  • a typical positive control could be an antibody recognizing a molecule which is known to be present in a sample to be tested.
  • a typical negative control could be an antibody recognizing a molecule which is known to be absent in a sample to be tested.
  • kits of parts for diagnosing Lyme disease in a subject comprising a) a source of Borrelia antigens and b) reagents for detecting the expression level of a pro-inflammatory cytokine.
  • the molecule which specifically binds a pro-inflammatory cytokine is an antibody.
  • a peptide-like molecule (referred to as peptidomimetic) or non-peptide molecule that specifically binds to a cytokine as defined herein and that may be applied in a method of the invention as defined herein may be identified using a method known in the art per se, as e.g. described in detail in US 6,180,084 .
  • Such a methods includes e.g. screening libraries of peptidomimetics, peptides, DNA or cDNA expression libraries, combinatorial chemistry and, particularly useful, phage display libraries. These libraries may be screened for an agonists and/or an antagonist of said cytokine by contacting the libraries with a substantially purified polypeptide of the invention, fragments thereof or structural analogues thereof.
  • Sequence identity is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences.
  • the identity between two amino acid or two nucleic acid sequences is preferably defined by assessing their identity within a whole SEQ ID NO as identified herein or part thereof. Part thereof may mean at least 50% of the length of the SEQ ID NO, or at least 60%, or at least 70%, or at least 80%, or at least 90%.
  • identity also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences.
  • similarity between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide.
  • Identity and similarity can be readily calculated by known methods, including but not limited to those described in ( Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988 ; Biocomputing: Informatics and Genome Projects, Smith, D.
  • Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include e.g. the GCG program package ( Devereux, J., et al., Nucleic Acids Research 12 (1): 387 (1984 )), BestFit, BLASTP, BLASTN, and FASTA ( Altschul, S. F. et al., J. Mol. Biol. 215:403-410 (1990 ).
  • the BLAST X program is publicly available from NCBI and other sources ( BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, MD 20894 ; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990 ).
  • the well-known Smith Waterman algorithm may also be used to determine identity.
  • Preferred parameters for polypeptide sequence comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970 ); Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992 ); Gap Penalty: 12; and Gap Length Penalty: 4.
  • a program useful with these parameters is publicly available as the "Ogap" program from Genetics Computer Group, located in Madison, WI. The aforementioned parameters are the default parameters for amino acid comparisons (along with no penalty for end gaps).
  • amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine.
  • Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.
  • Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place.
  • the amino acid change is conservative.
  • Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser or Ala; Gln to Asn; Glu to Asp; Gly to Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln or Glu; Met to Leu or Ile; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp or Phe; and Val to Ile or Leu.
  • Some aspects of the invention concern the use of an antibody or antibody-fragment that specifically binds a pro-inflammatory cytokine.
  • Methods for generating antibodies or antibody-fragments that specifically bind to a polypeptide are described in e.g. Harlow and Lane (1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY ) and WO 91/19818 ; WO 91/18989 ; WO 92/01047 ; WO 92/06204 ; WO 92/18619 ; and US 6,420,113 and references cited therein.
  • the term "specific binding,” as used herein, includes both low and high affinity specific binding.
  • Specific binding can be exhibited, e.g., by a low affinity antibody or antibody-fragment having a Kd of at least about 10 -4 M. Specific binding also can be exhibited by a high affinity antibody or antibody-fragment, for example, an antibody or antibody-fragment having a Kd of at least about of 10 -7 M, at least about 10 -8 M, at least about 10 -9 M, at least about 10 -10 M, or can have a Kd of at least about 10 -11 M or 10 -12 M or greater.
  • the verb "to comprise” and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.
  • the verb "to consist” may be replaced by "to consist essentially of” meaning that a method or an assay device as defined herein may comprise additional step(s), respectively component(s) than the ones specifically identified, said additional step(s), respectively component(s) not altering the unique characteristic of the invention.
  • reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements.
  • the indefinite article “a” or “an” thus usually means “at least one”.
  • the word "about” when used in association with an integer (about 10) preferably means that the value may be the given value of 10 more or less 1 of the value: about 10 preferably means from 9 to 11.
  • the word "about” when used in association with a numerical value (about 10.6) preferably means that the value may be the given value of 10.6 more or less 1% of the value 10.6.
  • PBMC Peripheral blood mononuclear cells
  • C healthy volunteers
  • P patients with Lyme disease
  • 1.10 6 cells/ml were stimulated in RPMI 1640 for 24h with the following strains of Borrelia:B. burgdorferi (ATCC 35210), B. garinii (ATCC 51383) and B. afzelii (ATCC 51567) that were first heat-inactivated by heating at 52°C for 30 minutes (1.10 5 /ml). Thereafter IL-1 ⁇ was determined by ELISA using antibody from R&D systems (IL-1F2). IL-1 ⁇ was strongly detected in Lyme patients (see figures 1, 2 ).
  • Blood (lithiumheparine) was taken from a subject suspected to have the Lyme disease. Blood was subsequently diluted in culture medium (1:5) and stimulated for 4 hours with 3 species of Borrelia common in Europe (see above). Subsequently, the supernatant was isolated via centrifugation and the presence of IL-1b was assessed by ELISA as described above.
  • PBMCs Peripheral blood mononuclear cells
  • IL-1 ⁇ or IFN- ⁇ Concentrations of human IL-1 ⁇ or IFN- ⁇ were determined using either specific or commercial ELISA kits (R&D Systems, Minneapolis or Pelikine Sanquin, Amsterdam, The Netherlands), in accordance with the manufacturers' instructions. Detection limits were 40 pg/mL for IL-1 ⁇ and for IFN- ⁇ ELISA (12 pg/mL).
  • Formaldehyde-inactivated cells were prepared by transferring or incubating them with 4% formaldehyde for one hour. Subsequently cells were washed several times with PBS . No stimulus was added to the control cultures. The cultures were incubated at 37 °C and 5% CO2 for 48 hours. After this incubation period, the supernatants were harvested and centrifuged at 15000g for 5 minutes, and thereafter stored at -20 °C until measurement of interferon ⁇ (IFN ⁇ ) and/or IL- ⁇ .
  • IFN ⁇ interferon ⁇
  • IFN ⁇ was measured using a specific ELISA (Pelikine Sanquin, Amsterdam, The Netherlands). IL-1 ⁇ was measured as described in example 1.

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Claims (9)

  1. Méthode non invasive pour diagnostiquer une maladie de Lyme chez un sujet, la méthode comprenant les étapes consistant à :
    (a) obtenir un échantillon du sujet qui est du sang, et facultativement diluer le sang, ou qui est dérivé du sang,
    (b) mettre en contact l'échantillon avec une source d'antigènes de Borrelia pendant 24 heures maximum,
    (c) déterminer le niveau d'expression d'une cytokine pro inflammatoire dans ledit échantillon à la fin de l'étape (b), dans lequel la cytokine pro-inflammatoire est IL-1β,
    (d) comparer ledit niveau d'expression à une valeur de référence, de préférence dans laquelle ladite valeur de référence est déterminée dans un échantillon témoin, et
    (e) diagnostiquer une maladie de Lyme chez ledit sujet lorsqu'un niveau d'expression détectable ou une augmentation du niveau d'expression de IL-1β est déterminé à l'étape (d).
  2. Méthode selon la revendication 1, dans laquelle le niveau d'expression d'une cytokine pro-inflammatoire supplémentaire sélectionnée parmi le groupe constitué de : IFNγ, IL-6 et IL-17 est avéré dans ledit échantillon à la fin de l'étape (b), dans laquelle une maladie de Lyme est diagnostiquée lorsque l'étape (c) aboutit à la découverte d'un niveau d'expression détectable ou d'une augmentation du niveau d'expression détectable de la cytokine pro-inflammatoire supplémentaire.
  3. Méthode selon l'une quelconque des revendications 1 ou 2, dans laquelle le niveau d'expression d'une cytokine pro-inflammatoire est déterminé en quantifiant directement la quantité de ladite cytokine pro-inflammatoire et/ou indirectement en quantifiant la quantité d'une séquence nucléotidique codant pour ladite cytokine pro-inflammatoire.
  4. Méthode selon l'une quelconque des revendications 1 à 3, dans laquelle ladite source d'antigène de Borrelia est une cellule de Borrelia entière, de préférence une cellule de Borrelia inactivée à la chaleur ou fixée par formaline est utilisée.
  5. Méthode selon l'une quelconque des revendications 1 à 3, dans laquelle ladite source d'antigène de Borrelia est un lysat d'une cellule de Borrelia
  6. Méthode selon l'une quelconque des revendications 1 à 4, dans laquelle l'échantillon est dérivé du sang du sujet.
  7. Méthode selon l'une quelconque des revendications 1 à 6, dans laquelle à l'étape (b) l'antigène de Borrelia provient d'une espèce de Borrelia sélectionnée parmi : B. burgdorferi, B. garinii et B. afzelii.
  8. Méthode selon l'une quelconque des revendications 1 à 7, dans laquelle à l'étape (b) si plusieurs espèces de Borrelia sont utilisées comme source d'antigènes de Borrelia, l'échantillon de l'étape (a) est divisé en plusieurs sous-échantillons, chaque sous-échantillon étant mis en contact avec une source d'antigènes provenant d'une espèce de Borrelia.
  9. Utilisation d'un dispositif d'essai dans une méthode telle que définie dans l'une quelconque des revendications 1 à 8, dans laquelle le dispositif comprend une molécule qui se lie de manière spécifique à la cytokine pro-inflammatoire IL-1β, de préférence dans laquelle la molécule qui se lie de manière spécifique à la cytokine pro-inflammatoire est un anticorps, et dans laquelle ledit dispositif est de préférence une bandelette d'essai à écoulement latéral.
EP11764357.7A 2010-09-21 2011-09-21 Nouveau procédé pour diagnostiquer la maladie de lyme en utilisant un test immunologique cellulaire Not-in-force EP2619584B1 (fr)

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US10274491B2 (en) 2014-07-07 2019-04-30 Veramarx, Inc. Biomarker signatures for lyme disease and methods of use thereof
WO2016134006A1 (fr) 2015-02-17 2016-08-25 Colorado State University Research Foundation Procédé à grande sensibilité pour la détection de la maladie de lyme précoce
KR102625783B1 (ko) 2015-09-25 2024-01-15 퀴아젠 사이언시스, 엘엘씨 라임병을 진단하기 위한 및 치료 후 라임병 스피로헤타 제거를 예측하기 위한 조성물 및 방법
WO2017119881A1 (fr) 2016-01-06 2017-07-13 Veramarx, Inc. Signatures de biomarqueurs pour la différenciation de la maladie de lyme et procédés d'utilisation associés
EP3399312A1 (fr) 2017-05-05 2018-11-07 Biomérieux Procédé de détection d'une réponse cellulaire immune
ES2933132T3 (es) 2017-05-05 2023-02-01 Biomerieux Sa Procedimiento para detectar una respuesta celular inmune
WO2018227109A1 (fr) 2017-06-08 2018-12-13 Colorado State University Research Foundation Différenciation de la maladie de lyme et de la maladie à éruptions cutanées associée aux tiques du sud
EP3456841A1 (fr) * 2017-09-15 2019-03-20 Centre National de la Recherche Scientifique (CNRS) Diagnostic et traitement de pathologies chroniques telles que la maladie de lyme

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9011861D0 (en) 1990-05-26 1990-07-18 Beecham Group Plc Novel compounds
US5723286A (en) 1990-06-20 1998-03-03 Affymax Technologies N.V. Peptide library and screening systems
GB9015198D0 (en) 1990-07-10 1990-08-29 Brien Caroline J O Binding substance
IL99552A0 (en) 1990-09-28 1992-08-18 Ixsys Inc Compositions containing procaryotic cells,a kit for the preparation of vectors useful for the coexpression of two or more dna sequences and methods for the use thereof
DK1471142T3 (da) 1991-04-10 2009-03-09 Scripps Research Inst Heterodimere receptor-biblioteker under anvendelse af fagemider
EP0686265A4 (fr) 1993-02-26 2001-08-22 Cambridge Biotech Corp Procedes induisant une immunite a l'arthrite de lyme et titrage colorimetrique concernant l'activite borreliacide d'antiserums
WO1998047343A2 (fr) 1997-04-04 1998-10-29 Biosite Diagnostics, Inc. Bibliotheques polyvalentes et polyclonales
US6180084B1 (en) 1998-08-25 2001-01-30 The Burnham Institute NGR receptor and methods of identifying tumor homing molecules that home to angiogenic vasculature using same

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
"AUTOMATION OF ELISA AND STRIP ASSAYS", INTERNET CITATION, 21 November 2014 (2014-11-21), XP007922907, Retrieved from the Internet <URL:http://www.bmgrp.com/fileadmin/user_upload/immunoassays/Biomedica_Borrelia_ELISA_-_Info_Leaflet.pdf> [retrieved on 20141121] *
B. GODBER ET AL: "Direct Quantification of Analyte Concentration by Resonant Acoustic Profiling", CLINICAL CHEMISTRY, vol. 51, no. 10, 1 October 2005 (2005-10-01), pages 1962 - 1972, XP055062522, ISSN: 0009-9147, DOI: 10.1373/clinchem.2005.053249 *

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DK2619584T3 (en) 2017-01-09
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US9316652B2 (en) 2016-04-19

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